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On the spontaneous aggregation of myosin
LETTERS
TO
THE
507
EDITORS
is a secondary effect of the deficiency. Since the amounts of these acids present in the necrotic area of the leaf were found to be much less than those in the green part adjacent to the necrosis, the chlorogenic and caffeic acids may be condensed by polyphenoloxidase to melanin pigments.
1. SPURR, A. R., Science 116, 421 (1952). 2. SWAIX, T., ~iochem. J. 63, 200 (1952). Znstitute for Atomic Research and Botany Zowa Slate College, Ames, Zowa Received July 24, 1956
On the Spontaneous
HAROLD J. PERKIXS S. ARONOFP
Department,
Aggregation
of Myosinl
At present there are in the literature some six values for the molecular weight of myosin ranging from 15 X 1Oj to 4 X 105. Laki and Carroll (1) have ascribed the differing values to the fact that myosin is altered considerably by exposure to room temperature. In the ultracentrifuge at 5°C. they observed a small leading peak produced a and a sharp major peak. Two hours’ exposure at room temperature single, very broad, asymmetric, more rapidly sedimenting peak. Whether this involves changes in shape, size, and/or aggregation of the molecules was not determined. We present here a brief account of experiments that help clarify this point.
a
b
FIG. 1. 0.36% myosin in 0.6 M KC1 at 25”C., centrifuged at 59,780 r.p.m. Fresh myosin; 51 min., (b) after standing 45 hr. at 25°C.; 46 min. 1 Contribution No. 1387 Sterling Haven, Connecticut.
Chemistry
Laboratory,
Yale University,
(a) New
508
LETTERS
TO
THE
EDITORS
Sedimentation and light-scattering measurements were made on myosin after exposure to room temperature for varying time intervals. Myosin was prepared by Szent-Gyorgyi’s (2) method, as modified by Mommaerts (3). The solvent used in the measurements was 0.6 df KCl. In the ultracentrifuge, at 25°C. and 0.36% protein, fresh myosin showed three peaks (Fig. 1): a sharp major peak (S~O,~ = 5.3) and two smaller peaks (SZO., = 5.8, 6.4). After 45 hr. at 25OC. the percentage of faster components had markedly increased, the slowest was almost gone, and the S 2D,Wvalues were 6.2,7.4, and 7.8. Intermediate standing intervals gave intermediate results. The light-scattering data are shown in Fig. 2 as angular envelopes extrapolated to zero concentration. The conventional interpretation gives the weight ,average molecular weight, M,, , and the r.m.s. radius of gyration, (&1’2 of the molecules, independently of their shape (4). These values are listed in Fig. 2. For a rod, (T)‘/* = 485 A corresponds to a length of 1680 A. The data show unambiguously that M, increases with exposure time at room
I
60
I
0.1
I
a2
I
03
I 0.4
I 0.5
I 0.6
I 0.7
I 0.6
I 0.9
I
sin* (8/z)
FIG. 2. Extrapolated light-scattering Fresh myosin, (b) after 22 hr. at 25”C., (c) retooled to 4°C. for 5-hr.
envelopes under various conditions. (a) (c) after 45 hr. at 25”C., (d) solution from
LETTERS
TO
THE
509
EDITORS
temperature, but (&I’2 stays constant. We therefore conclude that the molecules aggregate side to side, the three peaks being monomers, dimers, and trimers. The possibility of dissociating the aggregates was investigated. Neither nddition of ATl’ nor vcrsene had any effect. Raising the salt concentration to 2 JI or increasing t,he pH to 10 were also ineffective. Blum (5) has observed that acto~~/osin dissociates at pH 10 with no change in (j2)1’2. The absence of such dissociaiion here shows that the mechanism of aggregation is different in the tl7-o cases. Retooling t,o 4°C. for 5 hr. produced a small but, measureable dissociation (Fig. 2). It is not expected that longer periods at 4°C. would reverse it further. Indeed, aggregation occurs at 4”C., but at a slower rate. A sample of the fresh myosin kept cold for 16 days showed X, = 1.3 X 706, and (p-) I I12 = 485 A. Thus 16 da>-s at 4°C. are equivalent to 3-1 hr. at 25’C. It is clear that myosin aggregates side to side spontaneously and that the rate increases markedly with temperature. An!- discussion of the bearing this may have on the theory of muscle contraction must await the results of further studies on the “monomer” and the details of the aggregation. Such studies are in progress.
1. LAKI,Ti., AXD CARROLL, W. R.,Xalure 2. &EST-GY~RGYI, A., “The Chemistry
Academic
Press, New York,
176,389 (1955). of Muscular Contraction,”
2nd ed.
1951.
3. MOMMAERTS, W. F. H.&I.,.4ND PARRISH,R. G.,J. &Ol. Chm. 188, 545 (1951). 4. EHRLICH, G., AND DoTY,~?., J. AVL. Chem. Soc.?6,3764,App. II (1954). 5. BLUM, J. J., Arch. Biochenl. and Biophys. 43, 176 (1953).
Sterling Chemistry Laboratory, Department of Chemistry, Yale University, A’ew Haaen, Connecticut Receiced .Jzll!/ 24, 1956
Cysteine-Cystine
Content
ALFRED
and the Free Amino
Groups
HOLTZER
of Flagellid
Weibull showed that the flagella of Proteus vulgaris and Bacillus subtilis are proteinsceous, and reported that they contain <0.05’% cysteine and cystine (1,2). In another paper he reported that t,he flagella of the former organism contain one K-terminal alanine and 55 free e-amino groups (lysine)/105 g. of protein (3). The flagella of the above bact,eria give x-ray diffraction patterns characteristic of the keratin-myosin-epidermin-fibrinogen group of proteins, and the name “flagellin” has been suggested for the proteins of which flagella seem to be organized aggregates (4). We have been interested in the flagellins as models for fibrous proteins from bacteria (5-7), and therefore have compared the properties of flagellin isolated from various bacteria. Flngellins from mesophilic as well as thermophilic strains of bacteria were used in this comparison on the premise t,hat differences among 1 Supported Service.
in part
by a grant
(RG-4185(C))
from
the U. S. Public
Health
TO
THE
507
EDITORS
is a secondary effect of the deficiency. Since the amounts of these acids present in the necrotic area of the leaf were found to be much less than those in the green part adjacent to the necrosis, the chlorogenic and caffeic acids may be condensed by polyphenoloxidase to melanin pigments.
1. SPURR, A. R., Science 116, 421 (1952). 2. SWAIX, T., ~iochem. J. 63, 200 (1952). Znstitute for Atomic Research and Botany Zowa Slate College, Ames, Zowa Received July 24, 1956
On the Spontaneous
HAROLD J. PERKIXS S. ARONOFP
Department,
Aggregation
of Myosinl
At present there are in the literature some six values for the molecular weight of myosin ranging from 15 X 1Oj to 4 X 105. Laki and Carroll (1) have ascribed the differing values to the fact that myosin is altered considerably by exposure to room temperature. In the ultracentrifuge at 5°C. they observed a small leading peak produced a and a sharp major peak. Two hours’ exposure at room temperature single, very broad, asymmetric, more rapidly sedimenting peak. Whether this involves changes in shape, size, and/or aggregation of the molecules was not determined. We present here a brief account of experiments that help clarify this point.
a
b
FIG. 1. 0.36% myosin in 0.6 M KC1 at 25”C., centrifuged at 59,780 r.p.m. Fresh myosin; 51 min., (b) after standing 45 hr. at 25°C.; 46 min. 1 Contribution No. 1387 Sterling Haven, Connecticut.
Chemistry
Laboratory,
Yale University,
(a) New
508
LETTERS
TO
THE
EDITORS
Sedimentation and light-scattering measurements were made on myosin after exposure to room temperature for varying time intervals. Myosin was prepared by Szent-Gyorgyi’s (2) method, as modified by Mommaerts (3). The solvent used in the measurements was 0.6 df KCl. In the ultracentrifuge, at 25°C. and 0.36% protein, fresh myosin showed three peaks (Fig. 1): a sharp major peak (S~O,~ = 5.3) and two smaller peaks (SZO., = 5.8, 6.4). After 45 hr. at 25OC. the percentage of faster components had markedly increased, the slowest was almost gone, and the S 2D,Wvalues were 6.2,7.4, and 7.8. Intermediate standing intervals gave intermediate results. The light-scattering data are shown in Fig. 2 as angular envelopes extrapolated to zero concentration. The conventional interpretation gives the weight ,average molecular weight, M,, , and the r.m.s. radius of gyration, (&1’2 of the molecules, independently of their shape (4). These values are listed in Fig. 2. For a rod, (T)‘/* = 485 A corresponds to a length of 1680 A. The data show unambiguously that M, increases with exposure time at room
I
60
I
0.1
I
a2
I
03
I 0.4
I 0.5
I 0.6
I 0.7
I 0.6
I 0.9
I
sin* (8/z)
FIG. 2. Extrapolated light-scattering Fresh myosin, (b) after 22 hr. at 25”C., (c) retooled to 4°C. for 5-hr.
envelopes under various conditions. (a) (c) after 45 hr. at 25”C., (d) solution from
LETTERS
TO
THE
509
EDITORS
temperature, but (&I’2 stays constant. We therefore conclude that the molecules aggregate side to side, the three peaks being monomers, dimers, and trimers. The possibility of dissociating the aggregates was investigated. Neither nddition of ATl’ nor vcrsene had any effect. Raising the salt concentration to 2 JI or increasing t,he pH to 10 were also ineffective. Blum (5) has observed that acto~~/osin dissociates at pH 10 with no change in (j2)1’2. The absence of such dissociaiion here shows that the mechanism of aggregation is different in the tl7-o cases. Retooling t,o 4°C. for 5 hr. produced a small but, measureable dissociation (Fig. 2). It is not expected that longer periods at 4°C. would reverse it further. Indeed, aggregation occurs at 4”C., but at a slower rate. A sample of the fresh myosin kept cold for 16 days showed X, = 1.3 X 706, and (p-) I I12 = 485 A. Thus 16 da>-s at 4°C. are equivalent to 3-1 hr. at 25’C. It is clear that myosin aggregates side to side spontaneously and that the rate increases markedly with temperature. An!- discussion of the bearing this may have on the theory of muscle contraction must await the results of further studies on the “monomer” and the details of the aggregation. Such studies are in progress.
1. LAKI,Ti., AXD CARROLL, W. R.,Xalure 2. &EST-GY~RGYI, A., “The Chemistry
Academic
Press, New York,
176,389 (1955). of Muscular Contraction,”
2nd ed.
1951.
3. MOMMAERTS, W. F. H.&I.,.4ND PARRISH,R. G.,J. &Ol. Chm. 188, 545 (1951). 4. EHRLICH, G., AND DoTY,~?., J. AVL. Chem. Soc.?6,3764,App. II (1954). 5. BLUM, J. J., Arch. Biochenl. and Biophys. 43, 176 (1953).
Sterling Chemistry Laboratory, Department of Chemistry, Yale University, A’ew Haaen, Connecticut Receiced .Jzll!/ 24, 1956
Cysteine-Cystine
Content
ALFRED
and the Free Amino
Groups
HOLTZER
of Flagellid
Weibull showed that the flagella of Proteus vulgaris and Bacillus subtilis are proteinsceous, and reported that they contain <0.05’% cysteine and cystine (1,2). In another paper he reported that t,he flagella of the former organism contain one K-terminal alanine and 55 free e-amino groups (lysine)/105 g. of protein (3). The flagella of the above bact,eria give x-ray diffraction patterns characteristic of the keratin-myosin-epidermin-fibrinogen group of proteins, and the name “flagellin” has been suggested for the proteins of which flagella seem to be organized aggregates (4). We have been interested in the flagellins as models for fibrous proteins from bacteria (5-7), and therefore have compared the properties of flagellin isolated from various bacteria. Flngellins from mesophilic as well as thermophilic strains of bacteria were used in this comparison on the premise t,hat differences among 1 Supported Service.
in part
by a grant
(RG-4185(C))
from
the U. S. Public
Health